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This work investigated the effect of physiochemical conditions of fermentation on the de-emulsification capability of biological de-emulsifier production by a demulsifying strain (a Bacillus sp. isolated by our laboratory from oil contaminated soil). A quadratic orthogonal regressive rotation design was used to establish an effective statistical model for evaluation the combined effects of the incubation time(A), culturing temperature(B), glucose concentration(C) in substrate, inoculum age(D) and shaker agitation(E). The results of analysis of variance (ANOVA) of face-centered central composite design (FCCCD) showed that the adequately high R2 value of 0.976 and F-value of 30.69 indicated the statistical significance of the quadratic model which there were seven significant model terms (Prob>;F<;0.0500), namely B, C, E, AC, AE, C2, E2. De-emulsification capability was enhanced when the culturing temperature as a main influence factor was more than 35°C. The three-dimensional response surface plot and contour plot present the maximum demulsifying ratio was reached when the glucose concentration was at 8.5g/L~16.25g/L, the incubation time at the range of 15h~21h, the shaker agitation exceeded 150r/min and the inoculum age was at 16.5h~25.5h.